1. Field of the Invention
Nucleic acid hybridization has been employed for investigating the identity of nucleic acids. Hybridization is based on complementary base pairing. When single stranded nucleic acids are incubated in solution, complementary base sequences pair to form double stranded stable hybrid molecules. The ability of single stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with its complementary nucleic acid sequence has been employed as an analytical tool in recombinant DNA research. The availability of radioactive nucleotide triphosphates of high specific activity and the synthetic methods of incorporating these nucleotides into nucleic acid probes has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states mediated by alteration in nucleic acid composition of the host. These alterations in nucleic acid composition will include genetic or environmental change in DNA by insertions, deletions, point mutations, or acquiring foreign DNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine has not occurred because of the unavailability of a simple, sensitive, automated, nonisotopic, rapid method of DNA hybridization analysis.
Current methods for detecting DNA probes generally involve immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labelled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at elevated temperatures to remove unbound probe. The support is then dried and the hybridized material is detected by autoradiography or by colorimetric methods.
The current methods are slow and labor intensive. For that reason application of the current methods in clinical laboratories has not occurred. For such an application a simple, rapid, nonisotopic, homogeneous method for detecting DNA sequences is necessary.
2. Description of the Prior Art
Langer, et al., Proc. Natl. Acad. Sci. USA, (1981) 78, 6633-6637 discloses the enzymatic synthesis of biotin labelled polynucleotides and the use of these materials as novel nucleic acid affinity probes. The detection of viral genomes in cultured cells and paraffin imbedded tissue sections using biotin labelled hybridization probes is discussed by Brigati, et al., Virology, (1983) 126, 32-50. U.S. Pat. No. 4,486,539 discloses the detection of microbial nucleic acids by a one step sandwich hybridization test. Sensitive tests for malignancies based on DNA detection is described in U.S. Pat. No. 4,490,472. U.S. Pat. No. 4,480,040 discloses the sensitive and rapid diagnosis of plant viroid diseases and viruses employing radioactively labelled DNA that is complementary to the viroid or to the nucleic acid of the virus being diagnosed. European patent application No. 83106112.2 (Priority U.S. patent application No. 391,440 filed June 23, 1982) teaches modified labelled nucleotides and polynucleotides and methods of preparing, utilizing, and detecting the same. Methods and compositions for the detection and determination of cellular DNA are disclosed in U.S. Pat. No. 4,423,153.